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MOLECULAR BIOLOGY: WORKING WITH DNA

PROTEIN EXPRESSION: LABELING

Preparation of Radiolabeled 14C-Acetylated Collagen

Preparation of Radiolabeled 14C-Acetylated Collagen
 
Overview
This protocol prepares fibrillar collagen for in vitro Matrix Metalloproteinase (MMP)-1 assays by acetylating collagen with a 14C-acetyl group. MMPs are proteolytic enzymes capable of degrading connective tissue components such as collagen.
 
Procedure
All steps and reagents must be carried out at 4°C to prevent the formation of collagen fibers.

1. Pour approximately 50 ml of collagen into dialysis tubing and place in a beaker containing 500 ml of Borate Buffer (see Hint #1 and #2).

2. Dialyze the collagen against Borate Buffer for at least three consecutive days at 4(C, changing the buffer at least once a day.

3. Wash and bake for 1 hr a Hamilton syringe, a small beaker, and a stir bar.

4. Pour the dialyzed collagen into the small beaker and place in a larger container of ice.

5. While the collagen is stirring, add 1 mCi [14C] acetic anhydride (100 to 124 mCi/mmol) with the Hamilton syringe (CAUTION! see Hint #3)

6. Incubate 2 hr to label the collagen.

7. To remove unincorporated 14C label, dialyze the collagen against 0.1 M Acetic Acid 3 or 4 times at 4°C or until the dialysis buffer is no longer radioactive.

8. Dialyze the labeled collagen overnight at 4°C against TBS. The resulting collagen should contain between 1,000 to 2,000 cpm/ul.

Solutions
0.1 M Acetic Acid   Chill to 4°C before use.
Borate Buffer   200 mM CaCl2
Adjust pH to 9.0
Autoclave
10 mM Borate
Chill to 4°C before use.
TBS   25 mM Tris-Cl, pH 7.5
200 mM NaCl
Autoclave
Chill to 4°C before use.
 
BioReagents and Chemicals
Type I and type II Collagen
Borate
Tris
Sodium Chloride
Calcium Chloride
[14C] Acetic Anhydride
 
Protocol Hints
1. A mixture of bovine skin type I and type II collagens is sold by Celtrix under the trade name Vitrogen. It is mostly type I collagen and is supplied at a concentration of 3 mg/ml. Cellsystems sells a similar product called Cellon. Alternatively, see Protocol on Preparation of Fibrillar Collagen from Rat Tails to prepare collagen yourself.

2. The collagen will look slightly opaque at this stage.

3. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.